Methods and apparatus for injecting and sampling material through avian egg membranes

ABSTRACT

An injection apparatus for injecting and/or removing substances from subjects, such as avian eggs, includes an injection needle having a body portion and a tapered portion that terminates at a tip. A tube is attached to and extends around the injection needle in substantially concentric relationship therewith. The tube has an end portion positioned a predetermined distance from the needle tip. The tube end portion acts as a stop to limit the distance the needle tip can be inserted into a subject, such as an egg, through a membrane of the subject. A detector may be utilized to measure force exerted on a membrane of an egg by a stop attached to a needle. Force exerted on a membrane by a needle and/or by a needle and a stop attached to the needle may also be measured.

RELATED APPLICATION

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 60/618,110 filed Oct. 13, 2004, the disclosure ofwhich is incorporated herein by reference as if set forth in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to eggs and, more particularly,to devices for injecting and/or removing material from eggs.

BACKGROUND OF THE INVENTION

In poultry hatcheries and other egg processing facilities, eggs arehandled and processed in large numbers. The term “processing” includestreating live eggs with medications, nutrients, hormones and/or otherbeneficial substances while the embryos are still in the egg (i.e., inovo). In ovo injections of various substances into avian eggs have beenemployed to decrease post-hatch morbidity and mortality rates, increasethe potential growth rates or eventual size of the resulting bird, andeven to influence the gender determination of the embryo. Injection ofvaccines into live eggs have been effectively employed to immunize birdsin ovo. It is further desirable in the poultry industry to manipulate anembryo in ovo to introduce foreign nucleic acid molecules (i.e., tocreate a transgenic bird) or to introduce foreign cells (i.e., to createa chimeric bird) into the developing embryo.

In ovo injection of a virus may be utilized to propagate the particularvirus for use in preparation of vaccines. For some applications it maybe desirable to insert a sensing device inside an egg containing anembryo to collect information therefrom, for example, as described inU.S. Pat. No. 6,244,214 to Hebrank. In addition, it may be desirable toremove material samples from eggs, including embryonic andextra-embryonic materials.

Examples of in ovo treatment substances and methods of in ovo injection,as well as apparatus for handling a plurality of eggs, are described inU.S. Pat. No. 4,458,630 to Sharma et al. and U.S. Pat. No. 5,028,421 toFredericksen et al.

Many of the above-described in ovo methods require accurate positioningof a needle, sampling device or sensing device within an egg. However,it can be difficult to accurately and repetitively position a needle,sampling device, or sensing device within various compartments of an eggusing conventional in ovo apparatus and methods. Accordingly, there is aneed in the art for improved methods of accurately and repetitivelyplacing devices including, but not limited to, needles, sensing devicesand sampling devices within various compartments and locations of eggs.

SUMMARY OF THE INVENTION

In view of the above discussion, an injection apparatus for injectingand/or removing substances from subjects, such as avian eggs, isprovided and includes an injection needle having a body portion and atapered portion that terminates at a tip. A tube is attached to andextends around the injection needle in substantially concentricrelationship therewith. The tube has an end portion positioned apredetermined distance from the needle tip. The tube end portion acts asa stop to limit the distance the needle tip can be inserted into asubject, such as an egg, through a membrane of the subject. An annularspace is defined between the injection needle and the tube. Material,such as a sealant material is disposed within the annular space toprevent the flow of material through the annular space. Alternatively,the injection needle and tube may be fused together, thereby eliminatingan annular space.

According to other embodiments of the present invention, a disk ofmaterial (or other shapes of material) may surround an injection needleat a predetermined distance from the injection needle tip and serve thefunction of a stop when contact is made with a membrane or other tissueof a subject. The disk of material may be formed from various materialsand may have virtually any shape and configuration. An exemplary disk ofmaterial may be a bead of adhesive material applied circumferentially tothe injection needle.

According to other embodiments of the present invention, a detector(e.g., a weigh scale, load cell, etc.) may be utilized to measure theforce exerted on a membrane of an egg by a stop attached to a needle.Force exerted on a membrane by a needle and/or by a needle and a stopattached to the needle may also be measured.

According to other embodiments of the present invention, an apparatusincludes a tubular punch configured to form an opening in the shell ofan egg. A needle (e.g., an injection needle, sampling needle, etc.) ispositioned within the punch for movement therethrough and through anopening in an egg shell formed by the punch. A stop is attached to theneedle and is configured to contact the punch when the needle movesthrough the punch into an egg so as to limit the distance the needle isinserted within the egg.

According to other embodiments of the present invention, an apparatusincludes a tubular punch configured to form an opening in the shell ofan egg. A tubular locating member is reciprocally disposed within thepunch and a needle (e.g., an injection needle, sampling needle, etc.) isreciprocally disposed within the tubular locating member. The locatingmember is configured to be lowered into an egg through the opening andcontact a membrane (or other internal egg material) of an egg. Oncecontact is made, the locating member stops its downward motion and islocked into place so that it cannot move. The needle is then extendedinto the egg by a predetermined amount relative to the locating memberand a substance is injected into the egg and/or a sample of material istaken from the egg.

According to embodiments of the present invention, a force exerted bythe locating member on the membrane can be detected.

According to other embodiments of the present invention, a needle may beconfigured to act as a locating member. The needle has a blunt end thatis not sharp enough to rupture a membrane during the step of locatingthe membrane. Once contact with the membrane has been made, the needlecan the be pushed through the membrane with sufficient force and by apredetermined distance to inject a substance into the egg and/or removea sample of material from the egg.

According to other embodiments of the present invention, an apparatusfor sampling and/or injecting material from/into an avian egg includes atubular punch that is configured to form an opening in the shell of anegg, a tubular locating member reciprocally disposed within the punch,and a needle reciprocally disposed within the locating member. A lockingdevice is secured to the punch and is configured to lock the locatingmember in place relative to a membrane of an egg. The locking device isa compressible flange that has a tapered end portion configured tomatingly engage with the end portion of the apparatus housing. When theflange and housing are matingly engaged, the flange compresses aroundthe locating member and prevents movement of the locating memberrelative to the punch. The needle extends a predetermined distance pastthe locating member into an egg.

A method of injecting and/or sampling avian eggs, according toembodiments of the present invention, includes orienting an egg in apredetermined position; introducing an opening into the shell of theegg; extending a device through the opening in the egg shell; piercing amembrane within the egg via a needle tip and extending the needle intothe egg until a stop attached to the needle contacts the membrane.Material may be deposited and/or removed from the egg via the needle. Inaddition, information may be obtained from the interior of the egg via adetector associated with the device extended into the egg. The device isthen retracted from the egg.

A method of sampling material, such as allantoic fluid, from an egg,according to embodiments of the present invention, includes orienting anegg with the air cell up and forming an opening in the shell thereof. Alocating device is extended through the opening until contact with amembrane (e.g., the chorio-allantoic membrane (CAM)) is detected. Asampling device is extended from the locating device through the CAM bya predetermined amount and a sample of allantoic fluid is obtained viathe sampling device.

Embodiments of the present invention can be used to position a device atany location within an egg (e.g., the subgerminal cavity). The devicemay be utilized to deliver a substance and/or acquire a substance, andat any stage of embryo development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are side section views of injection apparatus, according toembodiments of the present invention.

FIG. 2 is a cross-sectional view of the injection apparatus of FIG. 1Ataken along line 2-2.

FIG. 3 is a cross-sectional view of an egg with the injection apparatusof FIG. 1A inserted therewithin and with the stop in contactingrelationship with a membrane.

FIG. 4A is a perspective view of an injection needle having a stopattached thereto, according to another embodiment of the presentinvention.

FIG. 4B is a cross-sectional view of an egg with the injection apparatusof FIG. 4A inserted therewithin and with the stop in contactingrelationship with a membrane.

FIG. 5 is a cross-sectional view of an egg with the injection apparatusof FIG. 1A inserted therewithin and with the egg in contactingrelationship with a force detector, according to other embodiments ofthe present invention.

FIGS. 6A-6C are side, cross-sectional views of an apparatus having alocating member reciprocally disposed within a punch and a needlereciprocally disposed within the locating member, according toembodiments of the present invention.

FIG. 7 is a flow chart that illustrates particular embodiments of thepresent invention.

FIGS. 8A-8B are side, cross-sectional views of an injection apparatushaving an injection needle with a stop, wherein the needle is surroundedby a tubular punch that is configured to form an opening in the shell ofan egg, wherein the punch is forming an opening in the egg shell in FIG.8A and wherein the injection needle is inserted within the egg in FIG.8B.

FIG. 9 is a flow chart that illustrates particular embodiments of thepresent invention.

FIGS. 10A-10F are side, cross-sectional views of an apparatus having alocating member reciprocally disposed within a punch and a needlereciprocally disposed within the locating member, according to otherembodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now is described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity. Broken lines illustrate optional features oroperations unless specified otherwise. All publications, patentapplications, patents, and other references mentioned herein areincorporated herein by reference in their entireties.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. As used herein, phrases such as “between X and Y” and“between about X and Y” should be interpreted to include X and Y. Asused herein, phrases such as “between about X and Y” mean “between aboutX and about Y.” As used herein, phrases such as “from about X to Y” mean“from about X to about Y.”

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting”, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of “over” and “under”. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

It will be understood that, although the terms “first”, “second”, etc.may be used herein to describe various elements, components, regions,layers and/or sections, these elements, components, regions, layersand/or sections should not be limited by these terms. These terms areonly used to distinguish one element, component, region, layer orsection from another element, component, region, layer or section. Thus,a “first” element, component, region, layer or section discussed belowcould also be termed a “second” element, component, region, layer orsection without departing from the teachings of the present invention.The sequence of operations (or steps) is not limited to the orderpresented in the claims or figures unless specifically indicatedotherwise.

The terms “avian” and “avian subjects,” as used herein, are intended toinclude males and females of any avian species, but are primarilyintended to encompass poultry which are commercially raised for eggs,meat or as pets. Accordingly, the terms “avian” and “avian subject” areparticularly intended to encompass various birds including, but notlimited to, chickens, turkeys, ducks, geese, quail, pheasant, parakeets,parrots, cockatoo, cockatiel, ostrich, emu, etc.

As used herein, the term “early embryo” refers to an avian embryo fromthe time of lay (blastodermal stage) through about the developmentalstage where primordial germ cells (PGCs) are migrating. With particularrespect to chicken embryos, an “early embryo” is generally about anembryonic stage 20 (H&H) embryo or earlier. The developmental stages ofthe chicken embryo are well-understood in the art, see e.g., The Atlasof Chick Development, R. Bellairs & M. Osmond, eds., Academic Press,1998, and need not be discussed further herein.

As used herein, the term “blastoderm” has its understood meaning in theart. Generally, a blastoderm includes an embryo from the time of laythrough the end of gastrulation. The blastoderm is sometimes referred toby the alternative designations “germinal disc” or “embryonic disc” inthe art. A blastoderm may be described as a flattened disc of cells thatforms during cleavage in the early embryo and persists until the end ofgastrulation. By the time of laying, two major regions of the blastodermare visible, the centrally-situated area pellucida and theperipherally-located area opaca (The Atlas of Chick Development, R.Bellairs & M. Osmond, eds., Academic Press, 1998). With particularrespect to chicken embryos, the blastoderm is typically characterized asan embryo from the time of lay (i.e., Stage IX or Stage X EG&K) throughabout stage XIII (EG&K) or higher.

As used herein, the terms “injection” and “injecting” encompass methodsof inserting a device into an egg or embryo, including methods ofdelivering or discharging a substance into an egg or embryo, methods ofremoving a substance (i.e., a sample) from an egg or embryo, and/ormethods of inserting a detector device into an egg or embryo.

The terms “chimeric bird” or “chimeric embryo” refer to a recipient birdor embryo, respectively, that contains cells (i.e., somatic cells and/orgametes) from another bird or embryo, referred to as a “donor.”

The terms “transgenic bird” and “transgenic embryo” are used herein inaccordance with their generally understood meanings in the art. Atransgenic bird or transgenic embryo contains a foreign nucleic acidsequence in one or more cells.

As used herein, the term “membrane” refers to any layer of tissue withinan egg that engages a stop attached to a needle, or other in ovo device,so as to prevent the needle from being inserted beyond a desiredlocation within the egg. Exemplary membranes within an egg include, butare not limited to, the outer shell membrane, inner shell membrane, thechorio-allantoic membrane, vitelline (VM) membrane, and amnioticmembrane (amnion).

Referring now to FIG. 1A, an injection apparatus 10 for injecting and/orremoving a substance from subjects, such as avian eggs, according toembodiments of the present invention, is illustrated. The illustratedinjection apparatus 10 includes an injection needle 12 having a bodyportion 14 and a tapered portion 12 a that terminates at tip 12 b. Tip12 b may be blunt or may have an aperture therein in communication withthe needle lumen (not illustrated). Needles and lumens within needlesare well understood by those skilled in the art of the presentinvention. If tip 12 b is blunt, an aperture is formed in the side ofthe needle adjacent the tip and is in communication with the needlelumen.

A tube 16 is attached to and extends around the injection needle 12 insubstantially concentric relationship therewith. The tube 16 has an endportion 16 a positioned a predetermined distance from the needle tip 12b. The tube end portion 16 a acts as a stop to limit the distance theneedle tip 12 b can be inserted through a membrane or other tissue of asubject, such as an egg.

The tube 16 does not have to extend completely around the needle 12.Tube 16 may only partially surround the needle 12, according toembodiments of the present invention.

FIG. 1B illustrates an injection apparatus 10′ for injecting and/orremoving substances from subjects, such as avian eggs, according toother embodiments of the present invention. In FIG. 1B, the injectionneedle 12′ is a “pulled pipette” needle having a very slender bodyportion 14′, as illustrated.

The injection needles 12, 12′ illustrated in FIGS. 1A-1B may be attachedto a tube 16 in various ways as would be understood by those skilled inthe art. For example, a needle and surrounding tube may be adhesivelyattached to each other or may be fused together via any of various knownmethods. Embodiments of the present invention are not limited to anyparticular method of attaching injection needles 12, 12′ and tube 16.

The injection needles 12,12′ illustrated in FIGS. 1A-1B may be formedfrom various materials including, but not limited to, glass, metals, andpolymeric materials. Exemplary injection needles are available fromHumagen, Inc. Moreover, injection needles having various shapes, sizesand configurations may be utilized in accordance with embodiments of thepresent invention. Needles may also have blunt tips and one or moreapertures formed in the side thereof for injecting material into an eggor sampling material from an egg. Embodiments of the present inventionare not limited to the illustrated injection needles 12, 12′. The tube16 surrounding the injection needles 12, 12′ may also be formed fromvarious materials including, but not limited to, glass and metal.

In the illustrated embodiments of FIGS. 1A-1B, an annular space 20 isdefined between the injection needle 12, 12′ and the tube 16. A sealantmaterial 22 is disposed within the annular space 20 to prevent the flowof material through the annular space 20, as illustrated in FIG. 2.Various types of sealant materials known to those skilled in the art maybe utilized, without limitation. According to embodiments of the presentinvention, the tube 16 and needle 12, 12′ may be fused together suchthat no annular space exists.

According to an embodiment of the present invention, the injectionneedles 12, 12′ are disposed within a capillary tube 16 having anoutside diameter of about 1.98 mm and an inside diameter of about 1.2mm. However, according to other embodiments of the present invention,tube 16 may have various diameters, lengths, and other dimensions,without limitation.

FIG. 3 illustrates the injection apparatus 10 of FIG. 1A with the tip 12b of the injection needle 12 extended to a predetermined depth within anegg 1 as a result of the tube 16 acting as a stop and making contactwith a membrane 2 within the egg 1. The membrane 2 may be any of anumber of membranes located internally within an egg. The needle tip 12b may be positioned in any of various locations within an egg. Theillustrated location 3 in FIG. 3 is a blastoderm.

According to other embodiments of the present invention, a disk ofmaterial 30 may surround an injection needle 12 at a predetermineddistance from the injection needle tip 12 b and serve the function of astop, as illustrated in FIGS. 4A-4B. As illustrated in FIG. 4B, theinsertion of the needle 12 into an egg stops (e.g., the downwardmovement of the needle is halted) when the disk of material 30 contactsa membrane 2. The disk of material 30 may be formed from variousmaterials and may have virtually any shape and configuration. Moreover,a disk of material may be located virtually anywhere on a needle 12. Anexemplary disk of material may be a bead of adhesive material applied tothe injection needle 12 (e.g., circumferentially or partiallycircumferentially).

According to other embodiments of the present invention, a forcedetector 40 may be utilized to measure the force exerted on a membraneof an egg by a stop, by a needle, and/or by a needle and a stop, asillustrated in FIG. 5. In the illustrated embodiment, the force detector40 is a weigh scale positioned beneath an egg 1. When a stop (e.g., tube16) makes contact with a membrane 2 within an egg 1, the downward forceis measured on the weigh scale 50. An exemplary weigh scale is availablefrom Ohaus Corporation (Pine Brook, N.J.). In addition, the forcerequired for the needle 12 to penetrate the membrane 2 can be measuredby force detector 40. The measurement of this force may allow anoperator to detect a position of a needle, or other device, beinginserted within an egg.

Alternatively, a load cell may be utilized as a force detector 40. Aswould be understood by those skilled in the art, an amplification and/orfiltering circuit may be utilized in conjunction with a load cell toboost the output and remove any unwanted noise. An analog/digitalconverter (e.g., a USB LabJack U12 from LabJack Corporation, Lakewood,Colo.) could be utilized to acquire force signals at some rate and asoftware program (e.g., LabView 7.0 from National InstrumentsCorporation, Austin, Tex.), could be used to graph the force signalswith respect to time.

The use of a weigh scale/load cell can also be utilized to detect thepresence of an egg in an egg flat. For example, if a needle is loweredbut no force is recorded/detected, it will be known that no egg ispresent. Thus, injection of a substance is not performed via the needle.

Referring to FIGS. 6A-6B, an apparatus according to embodiments of thepresent invention may include a tubular punch 50 that is configured toform an opening in the shell of an egg 1. In the illustrated embodiment,a tubular locating member 60 is reciprocally disposed within punch 50and a needle 12 is reciprocally disposed within the locating member 60.In FIG. 6A, the punch forms an opening in the shell of an egg 1. In FIG.6B, the punch is retracted from the egg 1 and the locating member 60,which is reciprocally movable within the punch 50, moves down into theegg 1 until locating member 60 contacts a membrane 2. When contact ismade, the locating member 60 stops its downwardly movement into the egg.In FIG. 6C, the locating member 60 has been locked into place and theneedle 12 is extended a predetermined distance past the locating member60 into the egg. The locating member 60 can be locked in place via anyof a various number of ways. Exemplary injection needle-punchconfigurations, which may be utilized in conjunction with embodiments ofthe present invention, are described in U.S. Pat. Nos. 4,903,635;5,136,979; and RE 35,973.

FIG. 7 is a flow chart that illustrates particular embodiments of thepresent invention. Referring initially to Block 200, an avian egg isoriented in a predetermined position (e.g., with the blunt end of theegg in a generally upward position, or with the egg positioned sideways,etc.). The egg may be oriented in virtually any position. For example,the egg may be oriented in a horizontal or vertical position (withrespect to the long axis) or at an angle therefrom during the storageperiod. In particular embodiments, the egg may be stored in the sameorientation as used for injection/sampling. Further, the egg may be heldin a fixed position (e.g., within a device) in which both side-to-sidemovement and rotation around the long axis of the egg are restricted orprevented.

An opening is introduced into the shell of the egg (Block 210) via apunch or other device known to those skilled in the art. According toembodiments of the present invention, the surface of an egg, at leastaround the site of injection, may be sanitized to reduce microbial (orother) contamination (e.g., with an alcohol or other sanitizingsolution). However, sanitizing an egg, including the site of injection,is not required with respect to embodiments of the present invention.

A device is extended through the opening in the egg shell (Block 220). Amembrane (or other tissue) within the egg is pierced by an injectionneedle and the needle is extended into the egg until a stop attached tothe needle contacts the membrane which halts the downward movement ofthe needle into the egg (Block 222). Material is deposited and/orremoved from the egg via the injection needle (Block 230). Informationmay also be detected from the interior of the egg via a sensor (Block240). The device is then retracted from the egg (Block 250).

Alternatively, a locating member is extended through the opening in theegg shell until it contacts a membrane within the egg which stops thedownward movement of the location member (Block 224). The locatingmember is then locked in position. A needle, sampling device and/orsensor is then extended by a predetermined amount past the locatingmember to accurately position the needle, sampling device and/or sensorat a desired location. Material is deposited and/or removed from the egg(Block 230). Information may also be detected from the interior of theegg via a sensor (Block 240). The device (locating member and needle) isthen retracted from the egg (Block 250).

The methods described herein may be fully manual, fully automated, orsemi-automated. For example, the steps of egg preparation andpositioning the embryo may be more suited for manual procedures. Thesteps of introducing an opening in an egg shell and inserting aninjection apparatus, may be manual, but are preferably automated.

In some embodiments, a multi-site injection or sampling device may beused, for example, as described in U.S. Pat. No. 6,032,612. Otherexemplary delivery and/or sampling devices include those described inU.S. Pat. No. 5,136,979; U.S. Pat. Nos. 4,681,063 and 4,903,635; andU.S. Pat. Nos. 4,040,388, 4,469,047, and 4,593,646.

In a further embodiment, an injection apparatus further comprising adetector as described in U.S. Pat. No. 6,244,214 is used to collectinformation regarding the position of an embryo (e.g., blastoderm) orother portion or compartment of an egg prior to or concurrently withinjection into the egg (for the purposes of sampling and/or delivering asubstance into the egg or embryo).

Those skilled in the art will appreciate that methods of the presentinvention may be carried out on a plurality of eggs, e.g., in acommercial poultry operation.

Referring to FIGS. 8A-8B, an injection apparatus 10″ according toembodiments of the present invention includes a tubular punch 50configured to form an opening in the shell of an egg 1. An injectionneedle (or sampling device, detector, etc.) 12 is positioned within thepunch 50 and is configured for reciprocal movement therethrough andthrough an opening in an egg shell formed by the punch 50. A stop 30 isattached to the needle 12 and is configured to contact the upper end 50a of the punch 50 when the needle 12 moves through the punch 50 into anegg 1 so as to limit the distance the needle is inserted within the egg1. In FIG. 8A, the punch 50 forms an opening in the shell of an egg 1.In FIG. 8B, the punch 50 also serves as a locating member and extendsdown until contact is made with a membrane 2 whereupon downward movementof the punch is stopped. The punch may then be locked into place. Theneedle 12 moves down into the egg 1 until the stop 30 contacts the upperend 50 a of the punch 50. Thus, the needle 12 is extended apredetermined distance into the egg after contact is made with themembrane 2 by the punch/locating member.

In an exemplary embodiment, the present invention may be used toaccurately and repetitively deliver a substance to an egg containing anearly embryo (e.g., a blastoderm) or to deliver a substance to the earlyembryo itself, for example to the subgerminal cavity. Any substance maybe injected by embodiments of the present invention, including but notlimited to cells, transgenic cells, vaccines, polypeptides,growth-promoting agents, probiotic cultures such as competitiveexclusion media, antibiotics, heterologous nucleotide sequencesincluding gene transfer vectors, vitamins, and/or markers such as dyes,etc. The substances may be injected alone, or in combination (e.g.,antibiotics may be included with the delivery of other substances). Asanother illustrative example, a dye or other marker may be included withother substances to be delivered to provide a means of determiningwhether delivery was to the desired location.

The present invention may also be advantageously used to introduce anucleotide sequence of interest into a developing embryo (preferably,the nucleotide sequence is stably transformed into the embryonic cells),i.e., to create a transgenic bird (as defined above). In otherembodiments, the present invention may be used to introduce a foreign or“donor” cell into a recipient embryo (i.e., to create a chimeric embryoand, optionally, a chimeric bird, as defined above).

Referring to FIG. 9, a method of sampling material (e.g., allantoicfluid, etc.) from an egg, according to embodiments of the presentinvention, is illustrated. An egg is oriented with the air cell up(Block 300) and an opening is introduced into the shell thereof (Block310). Egg manipulation and the formation of openings in the shell of anegg are well known to those skilled in the art, and can be accomplishedin various ways according to embodiments of the present invention.

A locating device is extended through the opening in the egg shell untilcontact with a membrane (e.g., the chorio-allantoic membrane (CAM)) ismade (Block 320). Movement of the locating device is halted and thenpreferably locked in place via any of various known methods. Thelocating device may be housed within a punch that is utilized to form anopening within the egg shell. However, embodiments of the presentinvention are not limited to such a configuration. The locating devicemay have virtually any shape and configuration as long as it isconfigured to make contact with a membrane, such as the CAM, and toallow a sampling/injection/detection device to be extended therefrom.

Contact with the membrane is detected via force exerted on the membrane.As described above with respect to FIG. 5, a force detector may beutilized to measure the force exerted on the membrane by the locatingdevice. The force exerted on the membrane may be measured by a scale,load cell or any other force or pressure sensing device. Force fromcontact on the membrane should not exceed about 30 grams as the membranemay be ruptured with a force greater than about 30 grams.

According to embodiments of the present invention, a sampling device isextended from the locked locating device through the membrane by apredetermined amount (Block 330) and a sample of allantoic fluid isobtained via the sampling device (Block 340). The sampling device may bea needle configured to draw allantoic fluid from the egg, as would beunderstood by those skilled in the art. For example, the needle may havea blunt tip and an axially-extending lumen that terminates at anaperture formed within a portion of the needle adjacent the tip.Allantoic (or other) fluid can be drawn into the lumen via the apertureupon subjecting the lumen to vacuum. The blunt tip prevents the lumenfrom becoming clogged with material.

The distance that the sampling device is extended is typically betweenabout three millimeters and about five millimeters; however, otherdistances and ranges of distances are possible. According to embodimentsof the present invention, the sampling device may include a stop, asdescribed above, that limits the distance the sampling device can beinserted through the membrane. According to other embodiments of thepresent invention, an injection device or a detection device may beextended from the locked locating device into the egg.

Embodiments of the present invention are advantageous for obtaining anytype of fluid from avian eggs including, but not limited to, blood,amniotic fluid, and allantoic fluid.

Embodiments of the present invention are especially advantageous forobtaining allantoic fluid from avian eggs. With variations in flock,flock age, storage time and other factors, the location and integrity ofthe CAM can be extremely variable. Accordingly, obtaining a sample ofallantoic fluid via conventional methods wherein needles travel a setdepth will often result in poor and/or variable sampling accuracy.Embodiments of the present invention positively locate the CAM thusensuring accurate location of a sample needle within allantoic fluid, aswell as within other types of fluid, such as blood or amniotic fluid,etc.

Referring now to FIGS. 10A-10F, an apparatus 100 for sampling and/orinjecting material from/into an avian egg, according to embodiments ofthe present invention, is illustrated. The illustrated apparatus 100includes a tubular punch 50 that is configured to form an opening in theshell of an egg 1. In the illustrated embodiment, a tubular locatingmember 60 is reciprocally disposed within punch 50 and a needle 12 isreciprocally disposed within the locating member 60. A locking device102 is secured to the punch 50 and is configured to lock the locatingmember 60 in place relative to a membrane 2 of the egg 1. Theillustrated locking device 102 is a compressible flange that has atapered end portion 102 a that is configured to matingly engage withtapered end portion 104 a of a housing 104 of the apparatus 100. Whenthe flange 102 and housing 104 are matingly engaged, the flangecompresses around the locating member 60 and prevents movement of thelocating member 60 relative to the punch 50. Embodiments of the presentinvention are not limited to the illustrated flange 102 for locking thelocating member 60. Various types of devices may be utilized for lockingthe locating member 60 without limitation.

In FIG. 10A, the apparatus 100 is moving downwardly such that the punch50 forms an opening (FIG. 10B) in the shell of an egg 1. In FIG. 10C,the locating member 60, which is reciprocally movable within the punch50, moves down into the egg 1 until locating member 60 contacts amembrane 2. When contact is made, the locating member 60 stops itsdownwardly movement into the egg and is “free floating” on top of themembrane 2. The term “free floating” means that the locating member 60is resting on the membrane 2 and no other loads are being applied to thelocating member 60.

Still referring to FIG. 10C, the housing 104 also moves downwardly andengages with the flange 102 (FIG. 10D) to lock the locating member 60relative to the punch 50. The housing 104 is then moved upwardly asillustrated in FIG. 10E with the flange 102 engaged therewith. As such,the flange 102, punch 50 and locating member 60 are moved upwardly as aunit. When the housing has been raised a predetermined amount, asillustrated in FIG. 10F, the locating member 60 is no longer incontacting relationship with the membrane 2. The needle 12 is thenextended a predetermined distance past the locating member 60 into theegg. In the illustrated embodiment, a stop 106 is attached to thelocating member 60. The needle 12 also includes a stop 108 associatedtherewith. When the needle 12 is moved downwardly into the egg, theneedle stop 108 engages the locating member stop 106 which stops thedownward movement of the needle 12 The stops 106,108 are configured toallow the needle 12 to be inserted a predetermined amount into an egg.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

1. An apparatus, comprising: a needle comprising a tip; and a stopattached to the needle a predetermined distance from the needle tip,wherein the stop limits the distance the needle tip can be inserted intoa subject through a membrane of the subject.
 2. The apparatus of claim1, wherein the stop comprises a tube that surrounds the needle.
 3. Theapparatus of claim 2, wherein the tube surrounds the needle inconcentric relationship therewith.
 4. The apparatus of claim 1, whereinthe stop comprises a disk of material.
 5. The apparatus of claim 1,further comprising a detector configured to measure force exerted on amembrane of a subject by the stop.
 6. The apparatus of claim 1, furthercomprising a detector configured to measure force exerted on a membraneof a subject by the needle tip.
 7. The apparatus of claim 1, furthercomprising a detector configured to measure force exerted on a membraneof a subject by the needle tip and stop.
 8. An egg injection apparatus,comprising: an injection needle comprising a tip; and a tube attached toand extending around the injection needle in substantially concentricrelationship therewith, wherein the tube has an end portion positioned apredetermined distance from the needle tip, wherein the tube end portionacts as a stop to limit the distance the needle tip can be inserted intoan egg through a membrane of the egg.
 9. The apparatus of claim 8,wherein an annular space is defined between the injection needle and thetube and wherein a sealant is disposed within the annular space toprevent the flow of material through the annular space.
 10. Theapparatus of claim 8, wherein the tube is adhesively attached to theinjection needle.
 11. The apparatus of claim 8, wherein the tube isfused to the injection needle.
 12. The apparatus of claim 8, furthercomprising a detector configured to measure force exerted on an eggmembrane by the tube end portion.
 13. The apparatus of claim 8, furthercomprising a detector configured to measure force exerted on an eggmembrane by the needle tip.
 14. The apparatus of claim 8, furthercomprising a detector configured to measure force exerted on an eggmembrane by the needle tip and tube end portion.
 15. The apparatus ofclaim 8, further comprising a tubular punch configured to form anopening in the shell of an egg, and wherein the injection needle ispositioned within the punch for reciprocal movement therethrough andthrough an opening in an egg shell formed by the punch.
 16. Anapparatus, comprising: a tubular punch configured to form an opening inthe shell of an egg; a needle positioned within the punch for reciprocalmovement therethrough and through an opening in an egg shell formed bythe punch, wherein the needle comprises a tip configured to be insertedwithin an egg; and a stop attached to the needle a predetermineddistance from the needle tip, wherein the stop is configured to contactthe punch when the needle moves through the punch into an egg so as tolimit the distance the needle tip is inserted within the egg.
 17. Theapparatus of claim 16, wherein the stop comprises a disk of material.18. A method of inserting a device within an egg, comprising: orientingan avian egg in a predetermined position; introducing an opening into ashell of the egg; extending a device through the opening in the eggshell, wherein the device comprises: a needle comprising a tip; and astop attached to the needle at a predetermined distance from the needletip, wherein the stop limits the distance the needle tip can be insertedinto the egg through a membrane thereof; and piercing a membrane withthe injection needle tip and extending the needle tip into the egg untilthe stop contacts the membrane.
 19. The method of claim 18, wherein thedevice is a delivery device and the method further comprises the step,following the piercing step, of releasing a substance through thedelivery device and depositing the substance into the egg.
 20. Themethod of claim 18, wherein the device is a sampling device and themethod further comprises the step, following the piercing step, ofremoving a sample of material from the egg.
 21. The method of claim 18,wherein the device is a detector device and the method further comprisesthe step, following the piercing step, of detecting with the detectordevice information from the interior of the egg.
 22. The method of claim18, wherein the egg contains a blastoderm, and wherein the device isextended through the opening in the egg shell to a location in theblastoderm.
 23. The method of claim 18, wherein the stop comprises adisk of material.
 24. The method of claim 18, wherein the stop comprisesa tube attached to and extending around the needle in substantiallyconcentric relationship therewith, wherein the tube has an end portionpositioned a predetermined distance from the needle tip, wherein thetube end portion acts as a stop to limit the distance the needle tip canbe inserted into an egg through the egg membrane.
 25. The method ofclaim 18, wherein introducing an opening into a shell of the eggcomprises forming the opening with a tubular punch, and whereinextending a device through the opening in the egg shell comprises movingthe injection needle through the punch and then through the openingformed in the shell of the egg.
 26. A method of sampling fluid from anegg, wherein the egg includes an air cell, the method comprising:orienting an avian egg with the air cell up; introducing an opening intoa shell of the egg; extending a locating device through the opening inthe egg shell until contact with a membrane is detected; locking thelocating device in contacting relationship with the membrane; extendinga sampling device from the locating device through the membrane by apredetermined amount; and obtaining a sample of fluid via the samplingdevice.
 27. The method of claim 26, wherein the membrane is thechorio-allantoic membrane.
 28. The method of claim 26, wherein the fluidcomprises allantoic fluid.
 29. The method of claim 26, wherein the fluidcomprises amniotic fluid.
 30. The method of claim 26, wherein the fluidcomprises blood.
 31. The method of claim 26, wherein detecting contactwith the membrane comprises detecting a force exerted on the membrane bythe locating device.
 32. The method of claim 26, wherein extending thesampling device through the membrane comprises extending the samplingdevice between about two millimeters and about six millimeters.
 33. Themethod of claim 26, wherein the sampling device comprises a needlehaving a blunt tip and an axially-extending lumen that terminates at anaperture formed within a portion of the needle adjacent the tip, andwherein fluid is drawn into the lumen via the aperture upon subjectingthe lumen to vacuum.
 34. The method of claim 26, wherein the samplingdevice comprises a stop that limits the distance the sampling device canbe inserted through the membrane.
 35. A method of injecting materialinto an egg, comprising: introducing an opening into a shell of the egg;extending a locating device through the opening in the egg shell untilcontact with a membrane is detected; locking the locating device inrelationship with the membrane; extending an injection device throughthe membrane by a predetermined amount relative to the locked locatingdevice; and injecting a substance into the egg via the injection device.36. The method of claim 35, wherein detecting contact with the membranecomprises detecting a force exerted on the membrane by the locatingdevice.
 37. The method of claim 35, wherein locking the locating devicein relationship with the membrane comprises locking the locating devicein contacting relationship with the membrane.
 38. A method of samplingmaterial from an egg, comprising: introducing an opening into a shell ofthe egg; extending a locating device through the opening in the eggshell until contact with a membrane is detected; locking the locatingdevice in relationship with the membrane; extending a sampling devicethrough the membrane by a predetermined amount relative to the lockedlocating device; and sampling material from the egg via the samplingdevice.
 39. The method of claim 35, wherein detecting contact with themembrane comprises detecting a force exerted on the membrane by thelocating device.
 40. The method of claim 35, wherein locking thelocating device in relationship with the membrane comprises locking thelocating device in contacting relationship with the membrane.
 41. Anapparatus, comprising: a tubular punch configured to form an opening inthe shell of an egg; a tubular locating device positioned within thepunch for reciprocal movement therethrough and through an opening in anegg shell formed by the punch, wherein the locating device is configuredto contact a membrane within an egg; a locking device configured to lockthe locating device in relationship with the membrane; and a needlepositioned within the locating member for reciprocal movementtherethrough, wherein the needle comprises a tip configured to beinserted through a membrane within an egg by a predetermined amountrelative to the locked locating device.
 42. The apparatus of claim 41,wherein the locking device is configured to lock the locating device incontacting relationship with the membrane.
 43. The apparatus of claim41, wherein the locking device is attached to the punch.
 44. Theapparatus of claim 41, wherein the needle is configured to remove asample from the egg.
 45. The apparatus of claim 41, wherein the needleis configured to inject a substance into the egg.